Irreversible colloidal chanis with recognition sites

ABSTRACT

A collection of colloidal particles in the form of one or several chains, in which the chains are generated in an irreversible manner and have at least one recognition site for a species, the site being different from sites implicated in the linear organisation of the particles. The invention further relates to a method for production of the collection, particularly for detection and/or dosage of at least one species in a fluid and a surface element functionalised by a collection of colloidal chains and a hybridisation network including such a surface element.

The invention relates mainly to magnetic colloidal particles organizedin the form of permanent colloidal chains. It is also directed towardthe use of these chains for detecting and/or analyzing specific speciespresent in a fluid.

Methods for sorting and/or analyzing species contained in a liquidsample are already known. In general, they use either hybridizationarrays arranged on surfaces like DNA or protein “chips”, or microbeads,or a combination of these two approaches. However, as emerges from theanalysis below, these techniques have limitations.

In the “chips”, the biological ligands under consideration (DNA,oligonucleotides, proteins) are deposited or synthesized in situ inpredetermined “spots” on a surface. Each spot has a typical surface areaof 100 microns by 100 microns or less, which makes it possible to have alarge number of recognition sites on a limited surface area, andtherefore to carry out a large number of molecular analyses with a smallamount of sample and in a limited period of time. Such systems, and alsomeans for preparing them, are described, for example, in U.S. Pat. No.5,744,305 (Affymetrix), Schena et al., Science, 270, 467-470, 1995.However, these chips have a sensitivity which remains insufficient forcertain applications. What is more, their hybridization kinetics aresignificantly slowed down compared to a conventional hybridization insolution. Finally, they exhibit a lack of reproducibility. In this case,these disadvantages are based, to a large extent, on physicochemicalaspects of the chips and of the hybridization mechanisms (see, forexample, M. S. Shchepinov, S. C. Case-Green, E. M. Southern, NucleicAcid Res. 25, 1155-61 (1997)).

The second method considered consists in binding the analytes to betested, using a network of microspheres arranged on a surface. Accordingto this technique, microspheres bearing various functionalitiesaccessible at their surface can advantageously be prepared. However, theactive surface area of a sphere remains, of course, of the same order ofmagnitude as that which it occupies on the surface of the test device.Consequently, this method does not therefore make it possible to obtaina significant gain in terms of sensitivity.

Magentic microbeads have also been proposed for analyzingoligonucleotides in a microfluid channel. These microbeads areintroduced into a channel and retained at a site of said channel by alocalized magnetic field: a zone essentially made up of a compact stackof magnetic beads is thus formed. The liquid containing the species tobe analyzed is then made to circulate through this stack. Thehybridization of said species is detected by fluorescence. Due to thecirculation of the species, the kinetics are clearly more rapid thanwith the conventional DNA “chips”. Moreover, the system is recyclable,since, by eliminating the magnetic field, the beads become mobile again.However, the concentrations of analytes used to demonstrate theprinciple of the method are much greater than those really used inchips, which suggests that the sensitivity is low. This may inparticular be explained by the compact assembly of beads. The beadsclosest to the detector form a screen for the transmission of the lightto the others, and it is therefore only the beads closest to the surfacewhich effectively participate in the detection.

Finally, microspheres are also used for identifying or analyzingspecies, and in particular biological species, in arrangements differentfrom networks arranged on a surface or in a microfluid channel. They arein particular the techniques known as “magnetic sorting”, which can beused analytically or preparatively. In a very conventional method, aliquid containing the species to be analyzed (cells, DNA, proteins) isbrought into contact with magnetic particles. The current version ofthese magnetic systems consists in introducing into the initial solutionmagnetic beads bearing functions specific for the cells to be isolated.After binding, the beads, and the species which are attached thereto,are pelleted using a magnet, whereas the supernatant is removed. Thismethod is currently proposed essentially for the binary sorting ofconcentrated objects. It then requires an on-line analysis to producethe information. This magnetic method, which is simple to implement, hashowever two important limitations: incomplete selectivity and a purelybinary nature, which both make it necessary to repeat the procedureswhen pure species or species corresponding to several criteria aresought. The lack of selectivity is caused first of all by the drainingof the supernatant during the “sedimentation” of the particles, themoving beads displace with them part of the fluid and therefore thesurrounding biological objects. The problems of nonspecific adhesionmust then be taken into account, the force of magnetic pressurecontributing to strongly anchoring to the beads any object trapped inthe pellet.

For its part, the present invention aims to propose a novel tool fordiagnosing and/or for preparing, identifying, analyzing or assayingspecies in a liquid sample, which gives satisfactory results both interms of sensitivity, kinetics and reproducibility.

More precisely, a first subject of the present invention is an assemblyof colloidal particles in the form of one or more chains, characterizedin that said chains are organized irreversibly and have at least onerecognition site for a species, said site being different from theligands involved in the linear organization of said particles.

For the purpose of the invention, the term “chain of colloidalparticles” or, without distinction, “colloidal thread” or “colloidalchain” is intended to mean an essentially linear assembly of colloidalparticles. Various geometric organizations of such assemblies can beused in the context of the invention. In particular, it is possible touse a “pearl necklace” assembly in which the width of the chain isessentially that of a colloidal particle, or a “column” assembly, inwhich each section of the chain comprises several particles.

The colloidal chains according to the invention have an aspect ratio(ratio of the length to the largest dimension of a cross section)significantly greater than 1, typically greater than 3, and preferablygreater than 5. For many applications, much higher aspect ratios, of 10or more, or even greater than 100, can however prove to be advantageous.

The colloidal chains according to the invention can be relatively rigid(adopting essentially the form of a rod), semi-rigid (capable of havinga radius of curvature comparable to their length), or flexible (capableof having a radius of curvature much smaller than their length). In thecase of flexible chains, the length in the description above extendsalong the curvilinear abscissa of said chain. According to a preferredvariant, they are semi-flexible or flexible.

In general, the cross section of the colloidal chains according to theinvention is essentially circular. However, it may also have any othershape, provided that the largest dimension of this cross section remainssmaller than the longest length of the colloidal chain, by a factor ofat least 3.

For certain applications, it is possible to use a single colloidal chainof a given type, by analogy to that which is already used for individualmolecules of DNA, in “single molecule” techniques. However, it isgenerally preferable to use a set of colloidal chains.

For the purpose of the invention, the term “colloidal particle” isintended to mean a compact three-dimensional object consisting of amultitude of atoms or of molecules, and capable of being maintained insuspension in a fluid. The dimensions of a colloidal particle aretypically between a few tens of nanometers and a few microns, morerarely a few tens of microns. By way of example, latex spheres,microgels or magnetic beads of micron or submicron size, nanocrystals ormicrocrystals constitute colloidal particles according to the invention.Preferably, such particles are maintained in suspension by Brownianmovement. However, particles which produce sedimentation can also beconsidered as colloidal for the purpose of the invention, provided thatit is possible to resuspend them at the time of their use, for exampleby agitation or sonication.

The colloidal particles used to constitute the chains of colloidalparticles according to the invention are preferably essentiallyspherical in shape.

These colloidal particles can be organic, mineral or organomineral.According to a preferred variant, they are completely or partly organicin nature, and preferably organomineral in nature, i.e. they have bothorganic constituents and mineral constituents.

Many types of organomineral particles are commercially available orknown to those skilled in the art. They advantageously make it possibleto combine properties derived from the organic portion and propertiesderived from the mineral portion, and therefore to construct colloidalchains according to the invention which have very diverse properties.

As regards the chemical nature of the mineral portion (or of the entireparticle if it is essentially mineral), it can also be very varied, andcan comprise in particular metal grains such as microparticles ornanoparticles of gold, of silver or of titanium, oxides ofsemiconducting metals, metal oxides, carbon particles, “quantum dots”with specific fluorescence or light absorption properties, and/ordielectric or conductive materials. Magnetic materials, such assuperparamagnetic, ferrimagnetic, ferromagnetic or antiferromagneticmaterials, or else conducting or semi-conducting materials, are mostparticularly suitable for the invention.

By way of oxides of semi-conductors, particles essentially consisting ofsilica or silicon oxide or comprising a silica shell are particularlyadvantageous.

This mineral portion can be either trapped at the heart of the colloidalparticles making up the colloidal chain, or present at their surface.For example, to obtain conduction properties, it is possible, accordingto a first preferred variant, to have a metal layer over the surface ofthe particles. Conveniently, this layer can be obtained by means of asilver-type depositing process, such as the many known by those skilledin the art (see, for example, DNA-templated assembly and electrodeattachment of a conducting silver wire, E. Braun, Y. Eichen, U. Sivan,G. Y. Ben-Yosph, Nature, 391, 775-778 (1998)). According to anotherpreferred variant, a significant fraction of metal or semi-conductinggrains may be included within the colloidal chain.

In the particular case of a magnetic material portion, it will bepreferred for said portion to be located at the heart of the colloidalparticles.

As regards the chemical nature of the organic portion, it can also bevery diverse, and can comprise in particular, by way of example, plant,petroleum-based or synthetic oils, various polymers such as derivativesof acrylamide, of polystyrene or of polycarbonate, which may or may notbe crosslinked, and, more generally, any of the materials used toconstitute latices.

In particular, suitable for the invention are colloidal particlescomprising a mineral core, coated with an organic layer of polymer type,such as, for example, polymers of polystyrene or polycarbonate type orderived from monomers of acrylic type, such as N-isopropylacrylamide,glycidyl acrylate or methacrylate, 2-hydroxyethyl methacrylate (HEMA) orethylene dimethacrylate (EDMA). It may also be poly(methylmethacrylate). An organic shell is particularly advantageous in so faras it offers, via the presence of its surface organic functions,possibilities of grafting for recognition sites and/or secondarycompounds and means for the linear organization of said particles. Anykinds of reactive functions, well known to those skilled in the art, canbe used as surface reactive functions. By way of nonlimiting example,they may be carboxylic, amine, alcohol or thiol functions, polymerizablefunctions such as double or triple bonds, in particular allyl or acrylicfunctions, or else polyols, hydrazines or epoxides. They may also beligands of biological type, such as biotin, streptavidin, avidin,digoxigenin or antidigoxigenin, and more generally antibodies orantigens commonly used as grafting sites in biology or else strongbinding sites for transition metals, such as “histidine cages” fornickel.

The assemblies of colloidal particles claimed are organized linearly soas to form an irreversible chain or a set of irreversible chains ofcolloidal particles.

For the purpose of the present invention, the term “irreversible” isintended to characterize the inability of the linear chains of thecolloidal particles to come apart spontaneously and/or after a briefperiod of time in the absence of an external field. In this case,excluded from the field of the invention are chains of particles forwhich the linear organization requires permanent maintenance of amagnetic or electric external field.

The irreversible nature of the assemblies of colloidal particlesaccording to the invention is, on the other hand, taken to mean undergiven conditions of composition of the fluid in which they aresuspended. Thus, such assemblies will be considered as irreversible evenif it is possible to dissolve them by diluting them in a liquid having acomposition or a pH significantly different from that of the liquid inwhich they were formed.

In the colloidal chains claimed, the cohesion between the particles can,in a preferred version, be maintained by covalent bonds between saidparticles, where appropriate resulting from bridging by means ofmolecules or macromolecules.

This covalent bond may involve specific interactions either directlybetween said particles or between the particles and molecules ormacromolecules, via reactive functions present at the surface of theseparticles. The reactive functions may be amine, carboxylic acid,alcohol, aldehyde, thiol, epoxide or hydrazine functions and/or halogenatoms.

The constituting and the maintaining of a linear organization betweenthe colloidal particles may also involve electrostatic, hydrophobic orVan der Waals interactions. To combine the colloidal particles with oneanother, it is also possible to involve specific interactions betweensaid particles, that are different from those exerted with respect tothe species to be analyzed or to be separated, either directly or bymeans of other molecules or macromolecules.

The assembly of particles claimed has at least one recognition site fora species and, preferably, several recognition sites of at least onegiven type.

The term “recognition site” is intended to mean a molecule, an ion, asurface element, or else a specific portion of a molecule or of an ion,capable of giving rise to an attractive interaction or to a chemicalreaction with a particular species or a particular category of species.

Several distinct types of recognition sites can be carried by the samechain or on distinct chains when a set of chains is used. The number oftypes of sites may in particular be greater than 5 or than 10, or even,in certain applications, such as for example DNA or protein “chips”,from several hundred to several tens of thousands.

The recognition sites characterizing the colloidal chains according tothe invention may be chosen, preferably, from nucleic acids (DNA, RNA,oligonucleotides), or synthetic analogs thereof (such as PNA, LNA,thiolated or methylated oligonucleotides), peptides, polypeptides,proteins, protein complexes, proteoglycans and polysaccharides. They mayalso be chosen from gene fragments, antibodies, antigens, enzymes orparts of enzymes, or biologically active parts of proteins, epitopes andhaptens. However, as specified above, the type of recognition siteconsidered for the purpose of detecting and/or assaying a species isdifferent from the specific ligands involved, for their part, in thepermanent organization of the colloidal particles in the form of a chainor chains. Thus, excluded from the field of the invention is a chain ofcolloidal particles in which the linear assembly is provided by thecovalent coupling of a pair of ligands like, for example, thebiotin/avidin pair, and which does not, moreover, have at least onerecognition site other than the specific ligands of the pair underconsideration, namely, in the example above, biotin or avidin.

The recognition sites present on the chains of particles claimed mayalso be chosen from chemical functions capable of specificallyrecognizing other chemical species, for example by bonding to them (forinstance, by way of example, crown ethers capable of bonding transitionmetals, or vice versa), or by reacting with them (for instance, still byway of example, trypsins or alpha-chymotrypsins, capable of digestingproteins). They may also consist of ligands specific for metals,molecular footprints, catalytic sites, hydrophobic groups or, moregenerally, the functionalities used in chromatography to give columns aspecific affinity for certain species. In particular, the recognitionsites present on the chains of particles claimed may be chosen fromcompounds comprising aromatic or heterocyclic chemical functions, orsites capable of giving rise to hydrogen bonds.

For the purpose of the invention, the term “species” is intended to meanmolecules or macromolecules, particles, atoms, ions, or objects ofnatural organic or artificial origin, such as nucleic acids, proteins,enzymes, antibodies, antigens, peptides, polypeptides, haptens,polysaccharides, proteoglycans, organelles, viruses, cells, sets ofcells, microorganisms or colloids. They may also be nanoparticles ormicroparticles of natural or artificial origin, organic or organomineralmolecules, drugs, medicinal products, or pollutants.

According to a preferred variant, a colloidal chain or a set ofcolloidal chains according to the invention has at least two distincttypes of recognition sites.

In this case, an entirely unique advantage of the colloidal chainsaccording to the invention is that, by virtue of their linear nature,they can have, along their backbone, various types of recognition sites.According to a preferred and very specific variant of the invention,these various types of sites are arranged in a predetermined (orsequenced) order along the colloidal chain(s) under consideration. Giventhe variety of accessible recognition sites, the ability to distinguishcolloidal chains having the same recognition sites in a different ordermakes it possible to have a much richer combination than theconventional colloidal particles, which cannot involve sequences.

In addition, compared to spherical particles, the colloidal chainsaccording to the invention have a better surface/volume ratio, at equalparticle volume. In this case, by attaching the colloidal chains to aflat surface and/or within a channel, a much greater active surface isprovided, compared to recognition sites deposited onto a surface, andthis active surface can in particular extend over several tens ofmicrons within said channel. This aspect of the invention is discussedin greater detail in the description hereinafter.

According to a preferred embodiment, the colloidal chains according tothe invention also have one or more labels, which may be identical ordifferent, that are especially useful for their detection.

Many labels of this type are known to those skilled in the art. Aparticularly advantageous family is that of the labels capable ofinteracting with electromagnetic radiation and, in particular, withvisible, ultraviolet or infrared light, or else capable of emittinglight under the action of a certain stimulus.

They may be labels capable of absorbing light within a certainwavelength range, or fluorescent or phosphorescent labels, such asmolecules, molecular complexes or “quantum dots”. It may also beadvantageous to use colloidal chains according to the invention whichhave molecules capable of electrochemical reactions (for instance, byway of example, hydroquinone and derivatives thereof), ofelectroluminescent effects or of chemiluminescent effects (electroactiveor chemoactive compounds). By way of example, a certain number ofhorseradish peroxidase-based luminescent labels are well known to thoseskilled in the art and can be used in the context of the invention.

Advantageously, the colloidal chains according to the invention, asopposed to the conventional colloidal particles, lend themselves to thebinding of one or more labels, which may be identical or different.

Of course, the embodiments described above can also apply to differentrecognition sites or to a combination of recognition sites and labels.

For certain applications, in particular for analyzing species orbiological fluids, it may be advantageous for the colloidal chainsaccording to the invention to also have on their surface moleculescapable of preventing nonspecific adsorption phenomena. Such moleculesare well known to those skilled in the art. They may in particular behydrophilic polymers such as polyoxyethylene, polypropylene glycol,polysaccharides and, in particular, dextran or else polyacrylamide, orhydrophilic polymers of acrylamide, such as “Duramide”,poly-N-acryloylaminopropanol, poly-N-acryloylamino-ethanol, polyvinylalcohol, polyvinylpyrrolidone, polydimethylacrylamide or copolymers ofdimethylacrylamide and of allyl glycidyl ether. Such polymers can begrafted onto the surface of the colloidal chains according to theinvention, either during the preparation of the initial particles orafter the formation of said chains, using reactive functions integratedat the surface of said particles, or by direct adsorption.

In the context of the invention, the sets of colloidal chainscharacterized in that they are divided up into several colloidal chains,each chain having a given type of recognition site or of reactivefunction and, where appropriate, at least one given type of label, areparticularly advantageous.

According to the applications, it is possible to use sets of colloidalchains which are substantially identical in length or, on the contrary,different in length.

According to a preferred variant, the colloidal chains in said set havea polydispersity in terms of length of less than 1.5, and preferablyless than 1.2. The polydispersities are understood to be mass-averages.

According to another preferred variant, it is possible to use the lengthof the colloidal chains as a criterion for differentiation between twosubfamilies and, therefore, to use, within a set of colloidal chains,several subfamilies of colloidal chains having different lengths, andessentially without any overlap of the size distribution between thevarious subfamilies. Preferably, in this variant, a correlation isestablished between the size of a colloidal chain and the type(s) ofrecognition sites that it has.

A second subject of the invention is a method that is useful forpreparing an assembly of colloidal particles as claimed, characterizedin that it comprises at least:

-   -   assembling colloidal particles in the form of one or more linear        objects, and    -   bringing said objects into contact with at least one agent        capable of irreversibly bridging them.

According to a preferred variant, this bringing into contact consists inmigrating said agent in the vicinity of said objects.

In this case, the first step can be carried out by applying, transientlyor permanently, to the colloidal particles an electric or magneticfield.

Thus, it is possible to confer a dipolar moment on colloidal particlesin suspension, by means of an external field: the dipoles orientthemselves in the direction of the field, attract along the axis of thefield and repel in the perpendicular direction, thus constitutingcolumns or “pearl necklaces”. According to one variant, it is possibleto use a direct or alternating electric field, and particles which arein suspension in a medium and which exhibit an electric polarizabilitydifferent from that of said medium.

According to a preferred variant, it is also possible to use magneticparticles which are aligned in a magnetic field. In this variant,superparamagnetic particles are particularly advantageous.

It is particularly convenient to perform the alignment of the colloidalparticles, so as to constitute colloidal chains according to theinvention, within a microfluid cell, and preferably within a channel ora chamber having at least two essentially parallel faces.

As regards the direction of the field, various geometries are possible.To obtain colloidal chains of uniform length, it is advantageous for thefield serving to align the colloidal particles to be essentially uniformand perpendicular to said faces. To obtain very long colloidal chains,however, a configuration may be preferred in which the field is parallelto a direction in which the cavity within which the alignment isperformed is large in size. In particular, it is, in this case,advantageous for the field to be parallel to the axis of the channel inwhich the alignment is performed or perpendicular both to this axis andto the smallest dimension of its cross section, if it is aparallelepipedal channel. When the field is parallel to the axis of thechannel, it may be advantageous to adjust the density of colloidalparticles such that a section of said channel contains, on average, onlyone object according to the invention or less.

As regards the step aimed at making the alignment of colloidal particlesirreversible, various protocols and/or types of particles can beconsidered.

A first protocol consists in stabilizing superparamagnetic colloidalparticles with a bridging agent of polymer type, and in particular apolyelectrolyte, for example of the polyacrylic acid type. In theabsence of a magnetic field, or in the presence of a weak field, theparticles exhibit, over a short range, a steric repulsion due to thepolymer chains. For a magnetic field greater than a threshold field, themagnetic particles are pressed increasingly strongly against oneanother: some chains can cross this steric barrier, and can effect abridging between the particles, which renders their associationessentially irreversible or at least gives it a very long lifetime. Anadvantage of polyelectrolytes, besides their ability to interactstrongly with particles of opposite charge, is that they can be broughtinto contact with the colloidal chains by means of an electric field.

A second protocol involves the assembly of the particles in columns bymeans of an external field within a cell having a semi-permeable wall,and the diffusion, across this wall, of a chemical agent capable ofcrosslinking the particles to one another.

It is also possible to use particles which bind to one anotherirreversibly under the simple action of an external field, without itbeing necessary to involve adjuvant molecules. Examples of thisembodiment are given in Examples 8 and 9. This cohesion is interpretedas the result of hydrophobic interactions between the particles, whichcan become involved after crossing a barrier of repulsive potentialunder the action of the external field.

Finally, the alignment of the particles can be made irreversible usingelectrostatic interactions: it is possible, for example, to organizenegatively charged (for example carboxylated) magnetic particles intofilaments, in a magnetic field, and then to bring them into contact withpolycations (for example using an electric field moving them in theopposite direction) the polycations, for instance, by way of example,polylysine or polyhistidine, attach to the particles and bridge themirreversibly, performing a “charge inversion” which converts the anionicreversible chain into a cationic irreversible chain. Examples of suchembodiments are given in Example 12. The process can be repeated with apolyanion, such as polyacrylic or polyglutamic acid, which performs asecond charge inversion. According to a particularly preferred variant,this second charge inversion can be obtained with a nucleic acid, which,at the same time, will play the role of recognition site and willtherefore convert the simple irreversible colloidal chain into acolloidal chain according to the invention, i.e. having recognitionsites. An example of such an embodiment is given in Example 7.

According to another embodiment, the method claimed comprises at least:

-   -   mixing colloidal particles and/or grafting them with at least        one bridging agent or a bridging agent precursor,    -   assembling said colloidal particles in the form of one or more        linear objects, and    -   initiating the bridging between said particles maintained in a        linear organization.

In this case, it is possible to begin the process by constituting acolloidal chain, and then bridging the particles by means ofelectromagnetic radiation, for example in the context of a photochemicalreaction. Use may, for example, be made of 5-azidonaphthalene-1-sulfonylchloride, 4,4′-diazidostilbene-2,2′-disulfonic acid or, more generally,photoreactive crosslinking agents such as those described in the“Molecular Probes” catalog, chapter 5.3.

A photochemical reaction can also be used indirectly. For example, it ispossible to form a mixture of magnetic colloidal particles havingcarboxylic functions, chains of polyamine in neutral form (for example,polylysine at a pH greater than 10.2), and orthonitrobenzyl, or moregenerally compounds comprising nitro or nitroso groups (see, forexample, H. Morrisson, The chemistry of the Nitro and Nitroso groups,Feuer H. Ed., Interscience, New York, 1969, section I, chapter 4, or R.Bressauer, J. P. Paris, in Advances in Photochemistry, W. A. Noyes, G.S. Hammond, J. N. Pitts editors, Interscience, New York, 1963, p. 275,or else, R. W. Yip et al., J. Phys. Chem., 95, p. 6078 (1991) R53). Thelatter compounds, otherwise known as “proton cages”, are capable, underthe action of ultraviolet radiation, of isomerizing and releasing aproton, creating an increase in pH sufficient to convert the neutralpolyamine chain to polycation. The advantage of this method is that itmakes it possible to avoid any aggregation between particles while themedium has not been subjected to light, and therefore to have the timeto perform the mixing of the various constituents and to organize thecolloidal particles in chains, before causing the light to act, whichwill create the attraction between the polymers and said particles, andthe bridging of the latter in the desired linear configuration.

The bridging between colloidal particles can also be initiated by meansof a change in temperature and/or a modification of pH.

Finally, if the initial particles spontaneously exhibit a short-rangeattractive potential and a long-range repulsive potential, it ispossible to initiate the bridging by raising the external field(magnetic field for magnetic particles, electric field for dielectricparticles).

According to a particular embodiment, the method claimed can beimplemented in a microfluid cell comprising, besides a channel 1, inwhich the assembling of the colloidal particles or the functionalizationthereof is performed, one or more secondary feed channels.

The organization of the colloidal particles into a chain can be carriedout on particles having recognition sites and/or identical or differentlabels beforehand, as in Example 8 or, on the contrary, onnonfunctionalized particles, as in Examples 6 and 7. In the first case,the various particles having identical or different recognition sitesand/or identical or different markers are organized so as to constitutesaid linear objects in a predetermined order. In this second case, theparticles organized in linear chains are equipped with recognition sitesand, optionally, with labels after constitution of said objects.

It is possible, of course, to combine the two variants above, i.e. toassemble, in a first step, colloidal particles having certainrecognition sites and/or labels, and to add to these particles, in anorder which may or may not be predetermined, other recognition sitesand/or labels.

Be that as it may, the colloidal particles used for separating thecolloidal chains have of course the specificities discussed above.

A third subject of the invention is a surface element bearing a linearassembly of colloidal particles according to the invention. In thiscase, it is particularly advantageous to have a surface exhibiting, atpredefined sites, irreversibly assembled colloidal chains according tothe invention.

Advantageously, the extension of these colloidal chains above thesurface makes it possible to significantly increase, firstly, thespecific surface for recognition and, secondly, the volume ofsupernatant solution brought into contact with the targets. In thiscase, the active surface area of said colloidal chains is greater thanthe surface area of the surface element bearing said chains and,preferably, by a factor of at least 4.

For example, with colloidal chains 200 nanometers in diameter,1-micrometer equidistant, and 20 micrometers long, the specific surfacearea is 12 square micrometers per square micrometer of projectedsurface.

Preferably, the colloidal chains are attached to the surface by one oftheir ends. This attachment can be obtained by creating a covalent bondbetween the chains and the surface, by bridging with molecules ormacromolecules and/or by electrostatic, hydrophobic or Van derWaals-type interactions. Specific interactions, different from thoseexerted between the recognition sites and the species, can also beenvisioned.

By attaching the colloidal chains to the surface via one of their ends,a “colloidal brush” is formed. An example of such a surface is given inExample 5. This brush can be extended actively within the supernatantsolution, for example by applying a magnetic field perpendicular to thesurface of the “chip” if the chains comprise magnetic materials.

In the majority of applications, it is advantageous to bring the chainson said surface together in a multiplicity of distinct domains,preferably at predetermined positions on said surface. Preferably, thesurface comprises at least two distinct domains comprising colloidalchains having different recognition sites.

Various methods can be used to constitute surfaces bearing a linearassembly according to the invention.

Typically, these methods should comprise the following steps:

-   -   grafting recognition sites onto the colloidal chains or onto at        least some of the particles constituting said chains, and    -   attaching said colloidal chains to said surface.

Preferably, the step for attaching the colloidal chains to the surfaceis carried out in the presence of an external field capable of aligningsaid chains. For example, if the chains have an electric dipolar momentor electric polarizability, an electric field may be used to thiseffect. If they have a magnetic dipolar moment or magneticpolarizability, a magnetic field may be used.

According to a preferred variant, said field exhibits a multiplicity oflocal gradients, which direct the chains toward predetermined sites onthe surface.

By means of these external fields, it is also possible to obtainsurfaces on which the colloidal chains are rather orientedperpendicularly or are rather oriented parallel to the surface,depending on whether said field is rather perpendicular or ratherparallel to the surface.

Optionally, these methods may also comprise a step for incorporatinglabels into said chains.

The order in which these various steps are carried out can varyaccording to the convenience of implementation. In particular, thegrafting onto the surface can be prior to, simultaneously with, orsubsequent to the placing of the recognition sites, subsequent to,simultaneously with, or prior to the placing of the labels. The placingof the labels, when this option is chosen, may, for its part, besubsequent to, simultaneous with or prior to that of the recognitionsites.

Finally, it is to be noted that the assembling of the colloidalparticles in chains may be prior to or simultaneous with the graftingthereof onto the surface. When this is possible, the second option ispreferred, since it decreases the number of steps required for obtainingthe final surface.

A subject of the invention is also a hybridization network comprising asurface element bearing colloidal chains according to the invention.This network may be low density, medium density or high density. Thesehybridization networks deposited onto a surface are generally referredto as “DNA chips”, “oligonucleotide chips” or “protein chips”.

In this type of application, the chains are grouped into a multiplicityof distinct domains on the surface element under consideration.Preferably, said domains occupy predetermined or pinpointable positionson said surface. Also preferably, at least two distinct domains comprisecolloidal threads having different recognition sites. Preferably, thereis a multiplicity of distinct domains each having a distinct type ofrecognition site. In certain cases, however, it may be desired tointroduce a certain redundancy between the domains, for the purposes ofcontrolling and/or measuring the reproducibility.

The “chips” formed from colloidal chains according to the inventionexhibit many advantages compared to conventional chips: firstly, theirspecific surface area is increased, which increases the sensitivity, inparticular in the case of competition with nonspecific ligands. Thesample volume and therefore the number of species contained in thesample placed in the immediate proximity of the recognition sites isalso considerably increased, which increases the kinetics and thesensitivity. Finally, in the case of magnetic colloidal chains, or moregenerally of colloidal chains sensitive to an external field, it ispossible to agitate these colloidal chains with respect to thesurrounding medium, for example by subjecting them to an oscillatingexternal, magnetic or electric field, which makes it possible toaccelerate the hybridization kinetics.

The use of colloidal chains according to the invention, of the type ofthose sensitive to an external field, also makes it possible to obtainmore reproducible networks: in fact, the colloidal chains can becalibrated in length, and their physical self-organizational propertiesimpose a uniform and predefined distribution over the entire surface ofthe domain.

Moreover, since the grafting of the recognition sites onto the filamentsis carried out in batch, it can be controlled to a greater degree thanin the case of conventional depositing or “spotting”, and can be thesubject of a quality control before the depositing onto the surface.

A subject of the invention is also a microfluid cell or channel or amicrocontainer containing an assembly of colloidal particles accordingto the invention. The term “microfluid cell” is intended to mean adevice comprising a channel or a set of channels, one of the dimensionsof which is between 100 nm and 1 mm, and which allows the transport offluids.

According to a preferred variant, said chains are organized within thechannel into a multiplicity of distinct domains. According to anotherpreferred variant, which does not exclude the preceding variant, saidchains are attached to one of the faces of the channel.

Finally, the colloidal chains according to the invention can also beused in an affinity electrophoresis, electrochromatography orchromatography device, in particular in order to act therein as aseparation matrix. In this case, the analytes contained in a sample areintroduced into said device. These analytes are transported thereinwithin a channel, by means of a suitable field (pressure field forchromatography, electric field for electrophoresis orelectrochromatography). The various analytes interact differently withthe recognition sites present on the colloidal chains, and are thereforeretained or slowed down to a greater or lesser degree. It is thenpossible to detect, by means well known to those skilled in the art(such as fluorescence, UV absorption, refractometry, electrochemistry,etc.), the various analytes after or during their passage between thecolloidal chains. The differences in passage time provide informationregarding the different affinities of the analytes with the colloidalchains and, where appropriate, regarding their nature. This method isparticularly suitable for microfluid systems, by virtue of the mostcommon dimensions of the colloidal chains according to the invention,which are in the micron range.

In another series of applications, the colloidal chains according to theinvention can be used as “microreactors”. In this case, the recognitionsites are catalytic sites, which make it possible to activate reactionswith a very large surface/volume ratio. If the colloidal chains are usedin bulk, they can, for example, be recovered easily by centrifugation orby magnetic sorting, and thus offer a good compromise between dissolvedcatalysts, which provide very good dispersion but are difficult torecover, and solid catalysts, which are easy to recycle by washing butrelatively nondispersed. An example of an embodiment of a microreactorbased on colloidal chains according to the invention is given in Example13.

According to another particularly preferred variant, the claimedcolloidal chains used as microreactors are attached to a surface, whichmakes it possible to exchange the reagents and to collect the reactionproducts as easily as with a solid catalyst, but with a much greatermobility and dispersion of the recognition sites.

The colloidal chains according to the invention are also advantageousfor combinatorial chemistry applications. In the case in point,colloidal chains having enzymes as catalysts are particularlyadvantageous for combinatorial chemistry or diagnosis. Thus, it ispossible, by way of nonlimiting example, to graft chymotrypsin oralpha-chymotrypsin onto magnetic colloidal particles according to theprotocol described in Bilkova J., Chromatogr. A, 852, 141-149 (1999).These particles are then assembled by means of one of the methodsdescribed in Examples 1 to 8. It is also possible to first perform theassembling of the colloidal chains from microspheres ofpoly(HEMA-co-EDMA) type, to functionalize them with hydrazide, asdescribed in Bilkova J., Chromatogr. A, 852, 141-149 (1999), and then toassemble them into colloidal chains according to one of the protocolsdescribed in Examples 1 to 8, and to repeat the protocol in step 2.9 ofBilkova J., Chromatogr. A, 852, 141-149 (1999) in order to attach thechymotrypsin in an oriented manner. Colloidal chains capable ofdigesting proteins are thus obtained.

A subject of the invention is also a molecular recognitionmicrocontainer or device comprising colloidal chains according to theinvention or a surface bearing such colloidal chains.

In fact, more generally, the colloidal chains according to the inventionprove to be particularly advantageous for a large number ofapplications, such as the analysis, isolation and/or preparation ofspecies.

Thus, a subject of the present invention is also a method for diagnosingand/or for analyzing, separating, purifying, assaying or identifying exvivo at least one species, using at least one assembly of particles asclaimed.

The species under consideration are those identified above.

These diagnostic and/or analytical methods can in particular be carriedout according to the protocol comprising at least the steps consistingin:

-   -   a) using an assembly of colloidal particles within a channel or        a container;    -   b) bringing said assembly into contact with at least one species        to be detected, separated and/or assayed; and    -   c) using means for detecting the possible hybridization of the        species under consideration with said assembly.

Optionally, in the preparative applications, it may also be advantageousto recover the products which have interacted with the colloidal chainsor, conversely, those which have not interacted.

Also optionally, the process also comprises a washing step during whichcertain products contained in the sample and which have not hybridizedwith the chains are removed, or during which the products which havereacted with said chains are recovered.

For the purpose of the invention, the term “hybridization” is intendedto mean any interaction in which a species binds specifically with arecognition site.

In the case of the claimed method, the chains of colloidal particles canbe arranged in the form of distinct zones made up of several chainswithin a channel or a surface. In such a case, the zone containing saidcolloidal chains is generally crossed by a fluid containing at least onespecies to be analyzed.

The invention is particularly advantageous for this type of operation:in fact, during the washing, the colloidal chains can lie down and thusprovide very little resistance to the flow, allowing easy and rapidwashing. As soon as the flow is stopped, they can stand up again (thisstanding up can optionally be activated by a magnetic field, if thecolloidal chains are magnetic) and again occupy a large volume. It isthus possible to combine the ease of washing obtained with open tubes,with the site density obtained with gels, but without the highresistance to the flow which the latter exhibit.

Among the means used in step c), fluorescence, phosphorescence,chemiluminescence, light absorption, surface plasmon resonance orradioactivity may preferably be used. It is also possible to use amethod of detection employing a measurement of current, for example inone or more circuits included in the molecular recognition device, inthe vicinity of the colloidal chain. The latter variant is particularlysuitable for the case of current-conducting colloidal chains, or forcolloidal chains using recognition sites capable of resulting inproducts that are detectable by an electrochemical reaction or a cyclicamperometry method. Use may also be made of one or more elements thatare sensitive to the magnetic field or a change in magnetic field. Thelatter variant is particularly suitable for colloidal chains havingmagnetic properties.

As regards the detection, according to a first variant, the detectioncan be carried out in situ, within the channel or the container in whichthe hybridization, or more generally the interaction between certainspecies contained in a fluid and the recognition sites borne by thecolloidal chains according to the invention, is carried out. Accordingto a second variant, this detection can be carried out in anotherdevice, after the hybridization or interaction phase. In particular, usemay be made of hybridization networks in accordance with the invention,in a manner comparable to conventional “DNA chips” or “protein chips”,by initially carrying out the hybridization in a hybridization chamber,and then carrying out the detection in a chip reader. When it is desiredto simultaneously carry out a search for molecular recognition involvinga multiplicity of types of colloidal chains, said chains can beorganized in relatively compact (typically circular) zones or “spots”,or, on the other hand, in strips, within a channel or on a surface.

By way of nonlimiting example, the colloidal chains according to theinvention, and the various components and devices using these colloidalchains, can be used for diagnosis; the search for and/or preparation ofmolecules or macromolecules, particles, atoms, ions, objects of naturalorganic or artificial origin, such as biologically active species, forinstance nucleic acids, proteins, enzymes, antibodies, peptides,polypeptides, polysaccharides, proteoglycans, organelles, cancerouscells, rare cells, epithelial cells, endothelial cells, cells forprenatal diagnosis, GMOs, pathogenic cells, viruses, antibodies ormicroorganisms; the search for chemical active materials such as toxicproducts, drugs, or pollutants; the recognition of animal, plant ormicroorganism varieties; the detection of mutations; the search forallergies; genotyping; the search for genes involved in diseases; thesearch for and/or preparation of reaction products derived fromcombinatorial chemistry protocols.

The examples and figures below are given by way of nonlimitingillustration of the field of the invention.

FIG. 1 a: Example of flexible colloidal chains of the “pearl necklace”type, irreversibly bridged with polyacrylic acid, from magneticparticles of mean diameter 1.3 micrometers plus or minus 0.3micrometers, prepared according to the protocol described in Example 1.

FIG. 1 b: Example of rigid colloidal chains of the “column” type and ofsemi-flexible colloidal chains of the “pearl necklace” type,irreversibly bridged with polyacrylic acid, from magnetic particles ofdiameter 1.3 micrometers plus or minus 0.3 micrometers, preparedaccording to the protocol described in Example 1.

FIG. 1 c: Example of monodispersed semi-flexible colloidal chains of the“pearl necklace” type, 70 micrometers long, organized irreversibly,according to the protocol described in Example 2.

FIG. 2 a: Example of colloidal chains bridged with polylysine, preparedaccording to Example 3.

FIG. 2 b: Example of colloidal chains bridged with polylysine andattached to a surface by their end, prepared according to Example 4.

FIG. 3: Example of colloidal chains having DNA molecules: a/ chainshaving one DNA molecule per chain on average, prepared according toExample 6; b: colloidal chain having a uniform covering of “Phi X 174”DNA, prepared according to Example 7.

FIG. 4: Colloidal chains having antibodies: a/ colloidal chains having“anti-mouse” antibodies, prepared according to Example 8; b/ colloidalchains prepared from beads 1 micrometer in diameter, and havingstreptavidin, prepared according to Example 9a.

FIG. 5: Enzymatic protease activity of an assembly of colloidalparticles according to the invention, prepared according to Example 13and having trypsin recognition sites.

FIG. 6: Capture of erythrocyte cells having a biotin site, within amicrochannel comprising a surface bearing assemblies of colloidalparticles according to the invention having streptavidin recognitionsites, prepared according to Example 14.

EXAMPLE 1

Preparation of colloidal chains from particles 1.3 plus or minus 0.3micrometers in diameter, using polyelectrolytes in the presence of amagnetic field, in a macroscopic container (test tube).

a/ Magnetic beads consisting of an inverse emulsion of octane-basedferrofluid (Rhone Poulenc), stabilized in water with sodium dodecylsulfate, are prepared according to the protocol described in “Emulsions:theory and practice”, Becher, P., Rheinhold, New York, 1965). A particlesize of 1.3 micrometers plus or minus 0.3 micrometers is selected byfractionated crystallization, according to the protocol described inBibette, J. Colloid Interface Sci., 147, 474. (1991). According to onevariant, commercially available magnetic particles, such as thosedistributed by the companies Bangs Laboratoires, Estapor, Merck,Eurolab, Prolabo, Uptima or Polysciences, can be used directly.

b/ The emulsion is washed several times (at least 5 times) with asolution of Nonyl Phenol Ethoxylate or NP10 (Sigma Aldrich) at 0.1%. Thewashing is carried out conveniently by pooling the magnetic drops at thebottom of the container with a magnet, replacing the supernatant withthe washing solution, and vigorously agitating (sonication mayoptionally be used in the case of slight aggregation of the particles),after having withdrawn the magnet. The operation is repeated as manytimes as necessary. At the end of washing, an amount of NP10 solution toachieve a particle concentration of the order of 0.1% by volume isadded.

c/ A solution of polyacrylic acid or PAA (Sigma Aldrich, Mw=250 000) isadded in order to achieve a PAA concentration of 0.1%. The pH should beequal to 4. The mixture is left to incubate with gentle agitation.

d/ The test tube containing the sample is placed in a coil, and themagnetic field is gradually increased. Chains form, and the tube is leftto incubate under the field for about 5 to 15 min. When a thresholdfield, of the order of 10 mT, has been exceeded, the chains remainirreversibly assembled after elimination of the magnetic field. Theiraverage length and their diameter can be regulated by adjusting theamplitude of the magnetic field and the concentration of the magneticparticles. By way of example, the chains shown in FIG. 1 a were obtainedwith a field of 50 mT, and a particle concentration of 0.1% by volume(observation between slide and cover, in the absence of magnetic field,under a Zeiss Axiovert 100 microscope using an immersion objective 100×,1.3. Under these conditions, a coexistence between flexible chains of“pearl necklace” type (FIG. 1 a), semi-flexible chains of “pearlnecklace” type (FIG. 1 b, on the left) and rigid chains of “column” type(FIG. 1 b, on the right) is obtained. For lower particle concentrations,pearl necklaces are essentially obtained, and for higher concentrations,columns are essentially obtained. This method makes it possible toreadily obtain large amounts of colloidal chains according to theinvention. On the other hand, these chains are quite polydispersed.

EXAMPLE 2

Production of Monodispersed Colloidal Chains of Calibrated Length

The procedure is carried out as in Example 1 up until step c.

For step d, instead of using a test tube, the solution is introducedinto a channel having a uniform thickness of 100 micrometers, preparedby molding of polydimethylsiloxane according to Xia, Y. Xia, G. M.Whitesides, Angew. Chem. Int. Ed, 37, 550 (1998), and then a magneticfield of 50 mT is applied perpendicular to the thickness of the channel.After elimination of the magnetic field, semi-flexible chains of uniformlength equal to the thickness of the channel (70 micrometers) areobtained (see FIG. 1 c) (observation conditions identical to those ofExample 1).

EXAMPLE 3

Preparation of Colloidal Chains Bridged with Polylysine

a/ Magnetic particles are prepared according to a/ of Example 1.

b/ These particles are introduced into a channel prepared as in Example2, by pressurization or capillarity, at a concentration which can vary,as needed, between 0.1% and 20%. A magnetic field of the order of 50 mTis applied perpendicular to the thickness of the channel. Colloidalchains are then obtained. These chains are of the “pearl necklace” typeif the initial concentration of the suspension of magnetic beads is low(typically less than 5%), and of the “column” type if this initialconcentration is high.

c/ Poly-L-lysine (0.1% w/v solution, Sigma Aldrich) is introduced intothe channel by electrophoresis (for a volume fraction of beads of 2%,poly-L-lysine is introduced such that its concentration in the channelis 0.05 wt %). For this, two electrodes are provided in two reservoirslocated at the ends of the channel. The polylysine is introduced, in theform of a solution, into one of the reservoirs, and the electrodelocated in this reservoir is brought to a positive potential withrespect to that of the electrode located in the other reservoir, so asto maintain within the channel an electric field of a few V/cm.Irreversible colloidal chains such as those observed in FIG. 2 a areobtained.

EXAMPLE 4

Preparation of Colloidal Chains Attached by one of Their Ends to aSurface, Bridged with Polylysine (FIG. 2 b)

a/ Magnetic particles are prepared or obtained as in step a of Example1.

b/ The emulsion is washed several times (at least 5 times) with asolution of Nonyl Phenol Ethoxylate or NP10 (Sigma Aldrich) at 0.1%. Thewashing is carried out conveniently by pooling the magnetic drops at thebottom of the container with a magnet, replacing the supernatant withthe washing solution, and vigorously agitating (sonication mayoptionally be used in the case of slight aggregation of the particles),after having withdrawn the magnet. The operation is repeated as manytimes as necessary. At the end of washing, an amount of NP10 solution toachieve a particle concentration of the order of 5% by volume is added.

c/ The continuous phase is replaced with a mixture consisting of 0.1 wt% NP10 and 0.89 wt % poly-L-lysine. For this, the magnetic beads arepooled at the bottom of the tube using a magnet, and the supernatant isremoved and replaced with the desired mixture.

d/ The emulsion is introduced into a channel. A magnetic field of 50 mTis applied perpendicular to the thickness of the channel. Afterelimination of the magnetic field, a brush of calibrated chains attachedto the lower wall of the channel is obtained.

EXAMPLE 5

Preparation of Colloidal Chains Attached by One of Their Ends to aSurface, Coupled with Polydimethylacrylamide

a/ Magnetic particles are prepared or obtained as in step a of Example1.

b/ A channel of uniform thickness is prepared by molding ofpolydimethylsiloxane according to Xia, Y. Xia, G. M. Whitesides, Angew.Chem. Int. Ed, 37, 550 (1998). This channel is filled with a solution ofpolydimethylacrylamide at 0.15% by mass, and is left to incubate for 40min.

c/ The channel is rinsed with a solution of Triton X405 at 2.1 g/l, andis then filled with the suspension of magnetic particles prepared in a.

d/ The channel is placed at the center of a coil and, afterequilibration of the pressures at the ends of the channel in order toavoid parasite flows, a magnetic field sufficient to create columns (ofthe order of one to a few tens of mT) is applied perpendicular to thethickness of the channel. The field is maintained for one hour.

e/ After elimination of the magnetic field, a “brush” of colloidalchains attached to the lower wall of the channel is obtained.

EXAMPLE 6

Preparation of Colloidal Chains According to the Invention, Having OneMolecule of “Lambda Phage” DNA Per Chain on Average.

a/ Colloidal chains are prepared according to the protocol described inExample 1.

b/ A solution of poly-L-lysine (0.1% w/v solution, Sigma Aldrich) isadded so as to obtain in the mixture a poly-L-lysine concentration of0.002 w %. The mixture is left to incubate with gentle agitation atambient temperature for 40 min.

c/ When DNA is added to the suspension, it attaches at certain points ofthe surface of the colloidal chains, according to a mechanism probablycomparable to that used on flat surfaces (see, for example, “The worldof Microarrays, J. Boguslavs.ky, Drug Discovery and Development, S5-S32(2001)). The concentration of DNA on the columns can be greatly varied,by adjusting the concentration of the DNA added to the solution, and itssize. In FIG. 3 a, approximately one large molecule of DNA (lambdaphage, Amersham Pharmacia Biotech Inc) was attached per colloidal chain.The colloidal chains have a dark appearance and the DNAs labeled with afluorescent marker (YOYO-1, Molecular Probes; one molecule of YOYO perten base pairs) are light in appearance (measurement carried out byepifluorescence on a Zeiss Axiovert 100 microscope equipped with amercury lamp for excitation and a 100× objective).

EXAMPLE 7

Preparation of Colloidal Chains According to the Invention Having aUniform Covering of DNA Molecules of “PhiX 174” type, Bound to theColloidal Chains by Means of Polylysine

The procedure is carried out as in Example 6, but using DNA which isdifferent in nature and a different DNA concentration for step c. Amixture of short DNAs of “PhiX 174” type is used (φX 174 RF DNA/Hae IIIFragments; Gibco BRL). Ultimately, the concentration of DNA is 0.5 μg/mland that of the poly-L-lysine is 0.002 wt %. The colloidal chains arethen washed by pelleting them in a tube using a magnet, and replacingthe supernatant with a solution identical to that used in b of Example6. The observation conditions are the same as for Example 6, and resultin colloidal chains uniformly covered with DNA (FIG. 3 b).

EXAMPLE 8

Production of Colloidal Chains According to the Invention, of the“Column” Type, Attached to a Surface and Having “Anti-Mouse” RecognitionFunctions

a/ A microfluid device comprising a channel of uniform thickness isprepared by molding of polydimethylsiloxane according to Xia, Y. Xia, G.M. Whitesides, Angew. Chem. Int. Ed. 37, 550 (1998).

b/ Anti-mouse Uptibeads (0.3 μm; Uptima), at the concentration of theoriginal solution as sold by the manufacturer, are sonicated so as tobreak up the aggregates present in the initial sample, and introducedinto the channel of the microfluid device prepared in a. This device isitself placed at the center of a coil so as to create within the channelan essentially uniform magnetic field oriented according to itsthickness. For the observation, the device surrounded by its coil isplaced on a Zeiss Axiovert 100 microscope and visualized using a 100×,1.3. immersion objective and a Cohu CCD camera. A magnetic field of 50mTesla is applied. When the magnetic field is eliminated, the particlesremain grouped in the form of columns attached to the inner surface ofthe channel via one of their ends, and which can turn and orientthemselves randomly around their point of attachment (figure a).

EXAMPLE 9

Production, by Direct Bridging, of Colloidal Chains According to theInvention, of the “Column” Type, Having Streptavidin Functions

Streptavidin Uptibeads (0.88 μm), at the concentration of the originalsolution as sold by the manufacturer, are sonicated so as to break upthe aggregates present in the initial sample, and placed in a microfluidcell as described in Example 8. When the magnetic field is eliminated,the particles remain grouped in the form of columns (FIG. 4 b).

EXAMPLE 10

Production of Colloidal Chains from Magnetic Particles Prefunctionalizedwith Galactose Oxidase in an Oriented Manner

a/ Preparation of the Magnetic Particles

Particles of HEMA-co-EDMA are prepared by polymerization in emulsion,and then activated with hydrazine, according to the protocol describedin Horak et al., Biotechnol. Progr., 15 (1999).

b/ Preparation of Activated (Oxidized) Galactose Oxidase

A solution of galactose oxidase from Dactylium dendroides (350 IU)(Sigma Aldrich) is dissolved in 2.5 ml of 0.1 M acetate buffer, pH 5.5,containing 2 mM CuSO₄ and 1 mM of D-Fucose (Acros Organics, Geel,Belgium). 100 IU of catalase (Sigma Aldrich) are added. After incubationfor 10 min at 37° C. and for 15 min at 4° C., 250 μl of NaIO₄ are addedto the solution and agitated for 30 min at 4° C., so as to selectivelyactivate the glycoside chains of the galactose oxidase. The reaction isstopped by adding 30 μl of ethylene glycol, and the agitation iscontinued for 10 min. The low molecular mass components are removed byfiltration through a Sephadex G-25 column.

c/ Attachment of the Oxidized and Purified Galactose Oxidase to theMagnetic Particles

1.5 ml of solution of activated magnetic particles, prepared in a, areadded to the purified galactose oxidase solution, such that thegalactose oxidase is in excess with respect to the particle surface.Incubation is carried out for 24 h at 4° C. with agitation. The supportis then washed with 0.1M acetate buffer, pH 4, containing 0.5M NaCl. Thewashing is repeated several times with a 0.1M phosphate buffer, pH 6,containing 2 mM CuSO₄, until the enzymatic activity of the eluent is nolonger detectable (the oxidase activity is measured using a test basedon the oxidation of D-galactose, as described in Avidad et al., J. Biol.Chem., 237, 2736 (1962)). The hydrazine groups which have not reactedare then blocked by incubation of the particles in a solution of 0.2Macetaldehyde in 0.1M acetate buffer, pH 5.5, for 24 h. Finally, theparticles are equilibrated in a solution of 0.1M phosphate buffer, pH 6,containing 2 mM CuSO₄.

d/ Formation of the Colloidal Chains

Polyacrylic acid or PAA (Sigma Aldrich, Mw=250 000) is added to analiquot of the solution of particles prepared in c, in order to achievea concentration of PAA of 0.1%. The mixture is left to incubate withgentle agitation. The test tube containing the sample is placed in acoil and the magnetic field is gradually increased (N.B. If a coil whichmakes it possible to apply a sufficient magnetic field is available, theaddition of PAA may be omitted, which makes it possible to leave thegalactose oxidase functions more accessible and therefore to improve thecatalytic yield of the final colloidal chains). Chains form and the tubeis left to incubate under the field for about 15 min. After eliminationof the magnetic field, irreversible colloidal chains having oxidaseactivity, demonstrated by spectrophotometric measurement using a testbased on the oxidation of D-galactose, as described in Avidad et al., J.Biol. Chem., 237, 2736 (1962), are obtained.

EXAMPLE 11

Functionalization of Colloidal Chains with Galactose Oxidase

a/ Preparation of the Magnetic Particles

A solution of colloidal magnetic particles is prepared, for exampleparticles of HEMA-co-EDMA prepared according to Horak et al.,Biotechnol. Progr., 15 (1999), or Ademtech particles. Polyacrylic acidor PAA (Sigma Aldrich, Mw=250 000) is then added so as to achieve aconcentration of PAA of 0.1%. The mixture is left to incubate withgentle agitation. The test tube containing the sample is placed in acoil and the magnetic field is gradually increased. Chains form and thetube is left to incubate under the field for about 15 min. Afterelimination of the magnetic field, irreversible colloidal chains areobtained. (N.B. If a coil which makes it possible to apply a sufficientmagnetic field is available, the addition of PAA can be omitted, whichmakes it possible to leave the galactose oxidase functions moreaccessible and therefore to improve the catalytic yield of the finalcolloidal chains). They are then activated with hydrazine according tothe protocol described in Horak et al., Biotechnol. Progr., 15 (1999).

b/ Preparation of the Activated (Oxidized) Galactose Oxidase

A solution of galactose oxidase from Dactylium dendroides (350 IU)(Sigma Aldrich) is dissolved in 2.5 ml of 0.1 M acetate buffer, pH 5.5,containing 2 mM CuSO₄ and 1 mM of D-Fucose (Acros Organics, Geel,Belgium). 100 IU of catalase (Sigma Aldrich) are added. After incubationfor 10 minutes at 37° C. and for 15 min at 4° C., 250 μl of NaIO₄ areadded to the solution and agitated for 30 min at 4° C., so as toselectively activate the glycoside chains of the galactose oxidase. Thereaction is stopped by adding 30 μl of ethylene glycol, and theagitation is continued for 10 min. The low molecular mass components areremoved by filtration through a Sephadex G-25 column.

c/ Attachment of the Oxidized and Purified Galactose Oxidase to theColloidal Chains

1.5 ml of solution of activated magnetic particles, prepared in a, areadded to the purified galactose oxidase solution, such that thegalactose oxidase is in excess with respect to the particle surface.Incubation is carried out for 24 h at 4° C. with agitation. The supportis then washed with 0.1M acetate buffer, pH 4, containing 0.5M NaCl. Thewashing is repeated several times with a 0.1M phosphate buffer, pH 6,containing 2 mM CuSO₄, until the enzymatic activity of the eluent is nolonger detectable. The hydrazine groups which have not reacted are thenblocked by incubation of the particles in a solution of 0.2Macetaldehyde in 0.1M acetate buffer, pH 5.5, for 24 h. Finally, thecolloidal chains are equilibrated in a solution of 0.1M phosphatebuffer, pH 6, containing 2 mM CuSO₄. The oxidase activity isdemonstrated by spectrophotometric measurement using a test based on theoxidation of D-galactose, as described in Avidad et al., J. Biol. Chem.,237, 2736 (1962).

EXAMPLE 12

Preparation of Irreversible Columns of Magnetic Particles of the“Microreactor” Type, Having Trypsin Recognition Sites for the Digestionof Proteins

Chains of magnetic particles are prepared according to Example 1. Thechains are rinsed and then resuspended in a phosphate buffer, pH 7.3, towhich Nonyl Phenol has been added, in a proportion of 1 mg of magneticparticles in 400 microliters of buffer (solution A). The chains aresedimented carefully, keeping the magnet at least 2 cm from the tube.The trypsin is then immobilized according to a protocol derived fromthat described in the work by Greg T. Hermanson “BioconjugateTechniques” 1996, Academic Press, London.

Furthermore, a solution B of 30 mg of ethylene carbodiimide (EDC) in 500microliters of phosphate buffer, pH 7.3, is prepared.

A solution C of 5 mg of S-NHS(N-hydroxysuccinimide) in 400 microlitersof phosphate buffer, pH 7.3, is also prepared.

Finally, a solution D is prepared: 7.5 mg of TPCK trypsin are dissolvedin 50 microliters of phosphate buffer, pH 7, and 5 microliters of asolution of benzamidine at 16 micrograms per milliliter are added.Solution D is immediately added to solution A, without a magnetic fieldand while stirring gently with a Gilson pipette, the end of the tip ofwhich has been cut off so as to decrease the shear. Solution B is thenadded, followed by solution C, still with gentle stirring. The solutionis left to incubate for 3 hours and is then washed by means of 2 or 3exchanges of buffer with a phosphate buffer, pH 7.3, containing NonylPhenol, identical to that of solution A. For the sedimentations, theprocedure is carried out as described for the preparation of solution A.

The activity of the trypsin is measured using a calorimetric assayaccording to the protocol described in H. F. Gaertner and A. J.Puigserver, Enzyme Micro. Technol. 14, 150 (1992) and P. S. Gravet etal., Int. Biochem. 23, 1085 (1991).

A series of solutions of BAPNA (benzoylarginine p-nitroaniline HCl) atmolar concentrations of between 0.1 and 1 are used as substrate, in aTris buffer. The assemblies of particles having trypsin are introducedinto the solution. After incubation for one hour, the amount ofp-nitroaniline produced by the digestion reaction is measured byabsorption of the solution at 410 nm, using a UV-Vis spectrophotometer(Shimadzu UV(160A)). The stability of the activity over time is given inFIG. 5.

In a variant, it is also possible to use, in the same protocol, magneticbeads based on silicon dioxide (SiO₂) (Kisker). Essentially mineralirreversible assemblies of colloidal particles according to theinvention are then obtained. According to this variant, the attachmentof a trypsin- or streptavidin-type recognition site is carried out byactivation of the particles with glutaraldehyde.

EXAMPLE 13

Preparation of Colloidal Chains Having Streptavidin Functions forCapturing Red Blood Cells Labeled with Biotin.

a/ A brush of colloidal chains having streptavidin functions is preparedin a channel, as described in Example 9.

b/ Human red blood cells are labeled with biotin according to thefollowing protocol:

-   -   6 μl of blood are placed in 1 ml of 270 mOsmol PBS,    -   washing is carried out 3 times with a 0.1M carbonate/bicarbonate        buffer, pH=8.5 (centrifugation at 3000 rpm for 1 min),    -   the pellet is taken and 500 μl of a solution of NHS-PEG        3400-biotin at 0.4 mg/ml are added,    -   incubation is carried out for 30 min,    -   centrifugation is carried out at 3000 rpm for 1 min in order to        remove the supernatant, and 500 μl of PBS+0.5% BSA are added.

c/ The blood cells are then introduced into the channel and migratethrough the brush of chains by means of an electric field, as described,for example, in Doyle et al., Science, 295, (5563), 2237, (2002). Theyattach to the columns of magnetic particles, as is demonstrated in FIG.6.

1-44. (canceled)
 45. An assembly of colloidal particles in the form ofone or more chains, wherein said chains are organized irreversibly andhave at least one recognition site for a species, said site beingdifferent from the ligands involved in the linear organization of saidparticles.
 46. The assembly of colloidal particles as claimed in claim45, wherein the colloidal particles are essentially spherical in shape.47. The assembly as claimed in claim 45, wherein said chains areflexible or semi-flexible.
 48. The assembly as claimed in claims 45,wherein said chain has an aspect ratio of greater than 1, and preferablygreater than
 3. 49. The assembly as claimed in claim 45, wherein saidparticles are totally or partly organic in nature, and preferablyorganomineral in nature.
 50. The assembly as claimed in claims 45,wherein said particles are essentially mineral in nature.
 51. Theassembly as claimed in claim 50, wherein said particles essentiallyconsist of silica or comprise a silica shell.
 52. The assembly asclaimed in claim 45, wherein said particles are based on aferromagnetic, ferrimagnetic, antiferromagnetic, superparamagnetic,conducting or semi-conducting material.
 53. The assembly as claimed inclaim 45, characterized in that the colloidal particles comprise amineral core coated with a polymeric organic layer.
 54. The assembly asclaimed in claim 45, wherein the cohesion between said particles ismaintained by covalent bonds between said particles.
 55. The assembly asclaimed in claim 45, wherein the cohesion between the particles resultsfrom bridging by means of molecules or macromolecules.
 56. The assemblyas claimed in claim 54, wherein the cohesion between the particlesinvolves specific interactions directly between said particles or withmolecules or macromolecules, via reactive functions present at thesurface of said particles.
 57. The assembly as claimed in claim 56,wherein the reactive functions are amine, carboxylic acid, alcohol,aldehyde, thiol, epoxide or hydrazine functions and/or halogen atoms.58. The assembly as claimed in claim 55, wherein the cohesion betweensaid particles involves interactions of electrostatic, hydrophobic orVan der Waals type.
 59. The assembly as claimed in claim 45, wherein therecognition site(s) is (are) chosen from: nucleic acids or syntheticanalogs thereof, peptides, polypeptides or proteins, protein complexes,proteoglycans, polysaccharides, gene fragments, antibodies, antigens,enzymes, epitopes, haptens, chemical functions capable of specificallyrecognizing other chemical species, ligands specific for metals,catalystic sites, molecular footprints, hydrophobic groups, enzymes orparts of enzymes.
 60. The assembly as claimed in claim 45, wherein therecognition site(s) is (are) molecules, ions, surface elements, or elsespecific portions of a molecule or of an ion that are capable of givingrise to an attractive interaction or to a chemical reaction with aparticular species or a particular category of species.
 61. The assemblyas claimed in claim 60, wherein the recognition site(s) is (are) chosenfrom compounds comprising aromatic or heterocyclic chemical functions orsites capable of giving rise to hydrogen bonds.
 62. The assembly asclaimed in claim 45, wherein said particles are organized in the form ofa single chain or of a set of colloidal chains having at least twodistinct types of recognition sites.
 63. The assembly as claimed inclaim 45, wherein the various types of recognition sites, or offunctions, are organized in a predetermined order along the chain(s)under consideration.
 64. The assembly as claimed in claim 45, whereinsaid particles or some of them have one or more labels which may beidentical or different.
 65. The assembly as claimed in claim 45, whereinit consists of several chains, each chain having a given type ofrecognition site or of reactive functions and, where appropriate, atleast given type of label.
 66. A method that is useful for preparing anassembly of colloidal particles as claimed in claim 45, comprising thesteps consisting of at least: assembling colloidal particles in the formof one or more linear objects, and bringing said objects into contactwith at least one agent capable of irreversibly bridging them.
 67. Themethod as claimed in claim 66, wherein the bridging agent is chosen frompolymers, and preferably polyelectrolytes.
 68. The method as claimed inclaim 66, wherein the linear assembly of said particles is obtained bytransient or permanent action of a magnetic field or of an electricfield.
 69. The method as claimed in claim 66, wherein the organizationof said particles is carried out in a microfluid cell or within achannel or a chamber having at least two essentially parallel faces. 70.A method that is useful for forming an assembly of colloidal particlesas claimed in claim 45, comprising the steps consisting in at least:mixing colloidal particles and/or grafting them with at least onebridging agent or a bridging agent precursor, assembling said colloidalparticles in the form of one or more linear objects, and initiating thebridging between said particles maintained in a linear organization. 71.The method as claimed in claim 70, wherein the third step involves amodification of temperature, application of electromagnetic radiation ofan electric field or of a magnetic field, a change in pH and/or aphotochemical reaction.
 72. A method for diagnosing and/or foranalyzing, purifying, identifying, separating or assaying ex vivo atleast one species, using at least one assembly of colloidal particles asclaimed in claim
 45. 73. The method as claimed in claim 72, wherein thespecies are chosen from proteins, nucleic acids, synthetic equivalentsof nucleic acids, proteoglycans, haptens, enzymes, antibodies, antigens,synthetic macromolecules, pollutants, organelles, cells, viruses,microorganisms, nanoparticles or microparticles of natural or artificialorigin, organic or organomineral molecules, drugs and medicinalproducts.
 74. The method as claimed in claim 72, comprising the stepsconsisting in at least: using, within a channel or a container, anassembly of colloidal particles in the form of irreversible chains;bringing said assembly into contact with at least one species to beseparated, detected and/or assayed, and using means for detecting thepossible hybridization of the species under consideration with saidassembly.
 75. The method as claimed in claim 74, further comprising awashing step during which the products which have not hybridized withsaid chains are removed, or during which the products which have reactedwith said chains are recovered.
 76. The method as claimed in claim 74,wherein the chains of colloidal particles are arranged in the form ofseveral distinct zones, within a channel or on a surface.
 77. The methodas claimed in claim 76, wherein the zone containing said chains iscrossed by the fluid containing at least one species to be analyzed. 78.The use of an assembly of colloidal particles as claimed in claim 45, inan electrochromatography, affinity electrophoresis or chromatographydevice.
 79. The use of an assembly of colloidal particles as claimed inclaim 45, as microreactors.
 80. The use of an assembly of colloidalparticles as claimed in claim 79, wherein the recognition site attachedto said particles is a catalytic site.
 81. The use of an assembly ofcolloidal particles as claimed in claim 45, in combinatorial chemistry.82. A surface element bearing an assembly of colloidal particles asclaimed in claim
 45. 83. The surface element as claimed in claim 82,wherein the active surface area of said colloidal chains is greater thanthe surface area of the surface element bearing said chains.
 84. Thesurface element as claimed in claim 82, characterized in that saidchains are bound to said surface via one of their ends.
 85. The surfaceelement as claimed in claim 82, wherein the attachment of said chains tosaid surface is obtained by creating a covalent bond between the chainsand the surface, bridging by means of molecules or macromolecules,and/or by electrostatic interactions of hydrophobic or Van der Waalstype.
 86. The surface element as claimed in claims 82, wherein saidchains are assembled, on said surface, into at least two distinctdomains comprising colloidal chains having different recognition sites.87. A hybridization network comprising a surface element as claimed inclaim
 82. 88. A microfluid cell or channel comprising an assembly ofcolloidal particles as claimed in claim 45.